Early onset mitochondrial encephalo-cardiomyopathy due to isolated deficiency of ATP synthase is frequently caused by mutations in TMEM70 gene encoding enzyme-specific ancillary factor. Diminished ATP synthase results in low ATP production, elevated mitochondrial membrane potential and increased ROS production. To test whether the patient cells may react to metabolic disbalance by changes in oxidative phosphorylation system, we performed a quantitative analysis of respiratory chain complexes and intramitochondrial proteases involved in their turnover. SDS- and BN-PAGE Western blot analysis of fibroblasts from 10 patients with TMEM70 317-2A>G homozygous mutation showed a significant 82-89% decrease of ATP synthase and 50-162% increase of respiratory chain complex IV and 22-53% increase of complex III. The content of Lon protease, paraplegin and prohibitins 1 and 2 was not significantly changed. Whole genome expression profiling revealed a generalized upregulation of transcriptional activity, but did not show any consistent changes in mRNA levels of structural subunits, specific assembly factors of respiratory chain complexes, or in regulatory genes of mitochondrial biogenesis which would parallel the protein data. The mtDNA content in patient cells was also not changed. The results indicate involvement of posttranscriptional events in the adaptive regulation of mitochondrial biogenesis that allows for the compensatory increase of respiratory chain complexes III and IV in response to deficiency of ATP synthase.

• MeSH
• Fibroblasts metabolism   pathology   MeSH
• Humans MeSH
• Membrane Proteins genetics   MeSH
• RNA, Messenger genetics   metabolism   MeSH
• DNA, Mitochondrial metabolism   MeSH
• Mitochondrial Proteins genetics   MeSH
• Mitochondrial Proton-Translocating ATPases deficiency   metabolism   MeSH
• Mitochondria enzymology   genetics   MeSH
• Mutation genetics   MeSH
• Oxidative Phosphorylation MeSH
• Peptide Hydrolases metabolism   MeSH
• Electron Transport Complex III metabolism   MeSH
• Electron Transport Complex IV metabolism   MeSH
• Gene Expression Profiling MeSH
• Electron Transport genetics   MeSH
• Up-Regulation * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Reactive oxygen species (ROS) generated by NADPH oxidase (NOX) are crucial for tip growth of pollen tubes. However, the regulation of NOX activity in pollen tubes remains unknown. Using purified plasma membrane fractions from tobacco and olive pollen and tobacco BY-2 cells, we demonstrate that pollen NOX is activated by calcium ions and low abundant signaling phospholipids, such as phosphatidic acid and phosphatidylinositol 4,5-bisphosphate in vitro and in vivo. Our data also suggest possible synergism between Ca(2+) and phospholipid-mediated NOX activation in pollen. Rac/Rop small GTPases are also necessary for normal pollen tube growth and have been proposed to regulate ROS production in root hairs. We show here elevated ROS formation in pollen tubes overexpressing wild-type NtRac5 and constitutively active NtRac5, while overexpression of dominant-negative NtRac5 led to a decrease of ROS in pollen tubes. We also show that PA formed by distinct phospholipases D (PLD) is involved in pathways both upstream and downstream of NOX-mediated ROS generation and identify NtPLDδ as a PLD isoform acting in the ROS response pathway.

• MeSH
• Cell Membrane enzymology   metabolism   MeSH
• Gene Expression MeSH
• Phospholipids metabolism   MeSH
• Monomeric GTP-Binding Proteins metabolism   MeSH
• NADPH Oxidases metabolism   MeSH
• Olea enzymology   growth & development   physiology   MeSH
• Hydrogen Peroxide pharmacology   MeSH
• Protein Isoforms MeSH
• Pollen Tube enzymology   growth & development   MeSH
• rac GTP-Binding Proteins metabolism   MeSH
• Reactive Oxygen Species metabolism   MeSH
• Plant Proteins metabolism   MeSH
• Signal Transduction MeSH
• Nicotiana enzymology   growth & development   physiology   MeSH
• Calcium metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: To strengthen research and differential diagnostics of mitochondrial disorders, we constructed and validated an oligonucleotide microarray (h-MitoArray) allowing expression analysis of 1632 human genes involved in mitochondrial biology, cell cycle regulation, signal transduction and apoptosis. Using h-MitoArray we analyzed gene expression profiles in 9 control and 13 fibroblast cell lines from patients with F1Fo ATP synthase deficiency consisting of 2 patients with mt9205deltaTA microdeletion and a genetically heterogeneous group of 11 patients with not yet characterized nuclear defects. Analysing gene expression profiles, we attempted to classify patients into expected defect specific subgroups, and subsequently reveal group specific compensatory changes, identify potential phenotype causing pathways and define candidate disease causing genes. RESULTS: Molecular studies, in combination with unsupervised clustering methods, defined three subgroups of patient cell lines--M group with mtDNA mutation and N1 and N2 groups with nuclear defect. Comparison of expression profiles and functional annotation, gene enrichment and pathway analyses of differentially expressed genes revealed in the M group a transcription profile suggestive of synchronized suppression of mitochondrial biogenesis and G1/S arrest. The N1 group showed elevated expression of complex I and reduced expression of complexes III, V, and V-type ATP synthase subunit genes, reduced expression of genes involved in phosphorylation dependent signaling along MAPK, Jak-STAT, JNK, and p38 MAP kinase pathways, signs of activated apoptosis and oxidative stress resembling phenotype of premature senescent fibroblasts. No specific functionally meaningful changes, except of signs of activated apoptosis, were detected in the N2 group. Evaluation of individual gene expression profiles confirmed already known ATP6/ATP8 defect in patients from the M group and indicated several candidate disease causing genes for nuclear defects. CONCLUSION: Our analysis showed that deficiency in the ATP synthase protein complex amount is generally accompanied by only minor changes in expression of ATP synthase related genes. It also suggested that the site (mtDNA vs nuclear DNA) and the severity (ATP synthase content) of the underlying defect have diverse effects on cellular gene expression phenotypes, which warrants further investigation of cell cycle regulatory and signal transduction pathways in other OXPHOS disorders and related pharmacological models.

• MeSH
• Principal Component Analysis MeSH
• Cell Line MeSH
• Phenotype MeSH
• Fibroblasts MeSH
• Financing, Organized MeSH
• Genome, Mitochondrial MeSH
• Humans MeSH
• DNA, Mitochondrial genetics   MeSH
• Mitochondrial Diseases MeSH
• Mitochondrial Proton-Translocating ATPases genetics   deficiency   MeSH
• Models, Genetic MeSH
• Oligonucleotide Array Sequence Analysis methods   statistics & numerical data   MeSH
• Sequence Deletion MeSH
• Cluster Analysis MeSH
• Gene Expression Profiling methods   statistics & numerical data   MeSH
• Check Tag
• Humans MeSH